Each step of the Human Immunodeficiency Virus (HIV) replication cycle represents a potential target for therapeutic intervention. A critical step is integration of the viral DNA into the host genome by the HIV integrase. We have set up an in vitro retroviral DNA integration system using recombinant HIV integrase and oligonucleotides which correspond to the U5 end of HIV DNA. Three steps of the integration reaction can be analyzed: (i) nucleolytic cleavage which removes 2 nucleotides from the 3'ends of the double-stranded DNA ('3'-cleavage'1'3'-processing'), (ii) DNA strand transfer which couples the Joining of the viral DNA into the target DNA with cleavage of the target DNA at the site of insertion ('integration'1 'strand transfer'), and (iii) the reverse reaction ('disintegration'). Using these assays, we have identified several groups of drugs that inhibit effectively HIV-1 integrase including caffeic acid phenethyl ester (CAPE). flavones, and some DNA intercalators such as chloroquine and anthracyclines which are active at micromolar concentrations. Interestingly, inhibitors of DNA topoisomerases I and II are generally inactive. Our current effort is aimed at investigating structure-activity of flavone, CAPE, and lignan derivatives, as well as new classes of non-DNA binders which could be tested for integrase inhibition in vivo. Long Terminal Repeats (LTRs) are repeated DNA sequences at both ends of retroviral DNA. Besides their role as HIV integrase target, the LTRs are the promoter regions of HIV DNA. They contain well defined transcription regulatory elements which are critical for making new viral RNA and proteins. Because of our recent finding that DNA topoisomerase II has preferential sites within the promoter region of the human MYC proto- oncogene (Pommier et al, Cancer Res. 1992;52:3125-30) and because topoisomerase I activity appears to facilitate transcription, we have mapped the interactions of DNA topoisomerases with the LTR legions and studied the effects of various topoisomerase inhibitors on the LTRs.